A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

Human senses are specifically designed to recognize and understand the world that surrounds us. Even though we have five senses, vision alone is responsible for at least 30 % of the sensory input to our brain. The visual process is initiated in a highly specialized cell type, the photoreceptors. These are light-sensitive cells located in the retina, a layered nervous tissue situated at the back of the eye. Retinal degeneration diseases are a highly heterogeneous group of conditions that include mutations affecting the survival, maintenance and proper functioning of photoreceptors or the adjacent retinal pigment epithelium (RPE). Such mutations, alone or in combination with environmental factors, cause the loss of the affected cells, and therefore, impairment of the visual sense. Retinitis Pigmentosa and Age-related Macular Degeneration are typical examples of retinal degenerative diseases eventually leading to blindness. In the first one, rod photoreceptors degenerate and consequently also cone photoreceptors are lost. The second is characterized by malfunction and loss of both, RPE and photoreceptor cells. Many current therapeutic approaches for the treatment of retinal degenerative diseases focus on slowing down the progression of the disease, rather than restoring the visual function. Currently, new therapies with the potential to recover the visual signal are under development. Some of these therapeutic strategies have already reached clinical stages, including gene therapy or retinal prosthesis. However, gene therapy approaches require the presence of remaining photoreceptors and, furthermore, particular targeting of disease-related genes. Retinal prosthesis still require improvement in terms of long-term biocompatibility and relevant visual function recovery. An alternative strategy for vision restoration is cell replacement of the lost photoreceptors, which is potentially suitable for targeting late stages of retinal degeneration diseases, independently of the inherent cause of the disease. Human vision relies primarily on cone photoreceptors, which are the cells responsible for color and high acuity vision under daylight conditions. However, cones represent a minority of the photoreceptors within the retina, and so, due to the low availability of these cells, cone photoreceptor transplantation studies lag behind rod transplantation studies. Consequently, in this study, strategies to increase the numbers of cone photoreceptors within mouse embryonic stem cells (mESC)-derived retinal organoids, which represent a potential cell source for transplantation studies, were explored. In this regard, I manipulated developmental pathways known to be involved in retinal development, such as Notch signaling, through the addition of various compounds in the retinal organoid maturation media. However, early cone markers have not yet been definitively identified, complicating the detection and isolation of cone photoreceptor precursors within the organoids. Therefore, a new early cone-reporter mESC line was generated in the course of this study as a valuable tool with the potential to facilitate the development of novel cone photoreceptor replacement therapies. Equally important in the field of photoreceptor cell replacement is the understanding of how the transplanted donor cells interact with the host retina. Previous studies have shown that visual function improvement is possible after transplanting rod or cone-like photoreceptor precursors into the sub-retinal space of mouse models for retinal degeneration. For many years it has been assumed that the underlying mechanism for the observed vision improvement was the migration and structural integration of donor cells into the host outer nuclear layer, where they mature and establish synaptic connections with the host retinal circuitry. However, experiments performed in this study demonstrate, for the first time, that upon transplantation donor and host photoreceptors exchange cytoplasmic material rather than structurally integrate into the host outer nuclear layer. Furthermore, insights into the transferred cytoplasmic content are given, i.e. that mRNA, but not mitochondria are exchanged by donor and host photoreceptors. This novel way of photoreceptor-photoreceptor communication led to a paradigm change in the field of retinal transplantation, requiring a re-interpretation of former transplantation studies. In addition, the discovery of the material transfer phenomenon might serve as a starting point for the development of novel therapeutic strategies based on cell-cell support for the treatment of retinal degenerative diseases. This study generated new knowledge in two important topics related to the development of cell therapies for retinal degeneration diseases, including the development of tools for cone transplantation studies as well as elucidating the interaction between donor and host cells upon transplantation.

Photoreceptors are sensory neurons in our eyes’ retinas that convert light into electrochemical signals thus allowing us to see the world around us. Human retinas have two types of photoreceptors: rods important for night vision and cones important for high-acuity daylight vision. In some retinal diseases, photoreceptors degenerate leaving patients visually impaired or even blind. One of the promising therapeutic approaches is cell therapy, which acts by supporting surviving or by substituting lost photoreceptors by transplanting donor photoreceptors produced in vitro. Human induced pluripotent stem cells (hiPSCs) represent a favorable donor cell source for transplantation, as they are patient-specific and have the ability to self-renew. While photoreceptors isolated from human retinal organoids represent bona fide cells for cell therapy, long time needed for their production, which coincides with developmental time, hampers their clinical application. Another approach for hiPSC differentiation is by overexpressing different transcription factors (TFs), the so-called forward programming. Although proved fast and efficient in producing multiple neuronal cell types, efficient forward programming protocols for engineering photoreceptors were so far not established. Therefore, aim of my thesis was to find TFs that drive in vitro photoreceptor differentiation from hiPSCs and to establish a fast forward programming protocol for producing photoreceptors in high yields. To find TFs that drive photoreceptor differentiation, I performed a TF-library-on-library screening in a reporter hiPSC line. The reporter hiPSCs expressed fluorescent markers only if synthetic photoreceptor-specific promoters were activated, i.e. in case of photoreceptor differentiation. The specificity of the reporter was confirmed in human retinal organoids. For the screening, I transduced the reporter cell line with two lentiviral libraries: a biased one consisting of 16 TFs known from in vivo photoreceptor development and an unbiased one consisting of 1756 TFs. After overexpressing TFs, cells that activated photoreceptor promoters were fluorescently sorted and analyzed. As 80 % of the sorted cells were positive for photoreceptor-specific genes, the screening was highly specific. Furthermore, the screening identified TFs that I validated in the reporter cell line as single, double and triple combinations to find the most efficient one in driving photoreceptor differentiation. The double combination of OTX2 and NEUROD1, known players in photoreceptor development, activated the cone reporter in 10 % of the cells, while additional overexpression of GON4L increased the activation to 25 %. GON4L was never before associated with photoreceptor development showing that in vitro differentiation might be uncoupled from its in vivo counterpart. The cone differentiation efficiency was increased to 50% by treating the cells with AraC, a cell cycle inhibitor that removes all proliferating cells from the cultures. Whether the cell will activate the cone reporter depended on expression levels of individual TFs. Higher and unequal levels, with NEUROD1 having the highest expression, were favorable for obtaining cells with activated cone reporter. Thus, by producing monoclonal cell lines, I identified competent clones with differentiation efficiencies higher than that of a polyclonal cell line and going up to 58%. Except activating the cone reporter, the cone precursor-like cells differentiated from hiPSCs by overexpressing the three TFs OTX2, NEUROD1 and GON4L acquired neuronal morphology and expressed photoreceptor precursor markers. As precursors are the optimal developmental time point for transplantation studies, the engineered cells were transplanted into mouse model of cone degeneration to assess their possible therapeutic potential. Some of the transplanted cells survived in vivo in the subretinal space but did not show any maturation or integration into the remaining retinal circuitry. Thus, further maturation of the cells in vitro is needed before the transplantation. So far, cone precursor-like cells showed ability to mature in vitro when co-cultured with retinal pigment epithelial cells derived from hPSCs and when cultured in presence of additional previously published growth factors. Therefore, changing culturing conditions from stem cell to photoreceptor-specific showed beneficial for further in vitro maturation and paved the way for further research. In conclusion, this study advanced TF-mediated cone photoreceptor engineering. It showed that overexpressing the three TFs OTX2, NEUROD1 and GON4L is enough to push differentiation of more than 50% of hiPSCs into cone precursor-like cells in only 10 days. Additional research to improve maturity and homogeneity of engineered cells – overexpressing additional TFs and changing culturing conditions is ongoing. Fast and efficient protocol established in this study is beneficial for bringing in vitro differentiated cone photoreceptors closer to their commercial application. Such engineered cones could be used as biomedical testbeds for drug discovery and research and represent a promising donor material for cell transplantation to treat blindness.

Genetisch kodierte Photorezeptoren in Modellorganismen begründen die Optogenetik. Sie ermöglicht die nicht-invasive, reversible und räumlich-zeitlich präzise Perturbation von zellulären und physiologischen Signalprozessen durch Licht. Natürliche photoaktivierte Adenylylzyklasen (PACs) steigern die intrazelluläre Konzentration des Botenstoffs zyklischen Adenosinmonophosphats (cAMP) durch Blaulicht. Damit erlauben sie die optogenetische Analyse von cAMP-abhängigen Signalwegen. Diese Arbeit komplementiert PACs durch die synthetische rotlichtaktivierte Phosphodiesterase LAPD zur Degradation von cAMP und zyklischem Guanosinmonophosphat (cGMP). LAPD ist eine Chimäre aus dem photosensorischen Modul von Deinococcus radiodurans Bakteriophytochrom (DrBPhy) und der Effektordomäne der cAMP/cGMP-spezifischen H. sapiens Phosphodiesterase 2A (HsPDE2A). Die Fusionsstelle wurde von den helikalen Linkern zwischen Sensor- und Effektormodulen durch strukturelle Überlagerung abgeleitet. LAPD inkorporierte den Chromophor Biliverdin (BV) nach Expression in E. coli und Reinigung vollständig und entsprach spektral und photochemisch dem Wildtyp-DrBPhy. Durch Bestrahlung mit Rot- und Fernrotlicht (R bzw. FR) wurde LAPD in die metastabilen photochemischen Zustände Pfr (fernrot) bzw. Pr (rot) umgewandelt. Vollständig aktivierte LAPD katalysierte die Hydrolyse von cGMP und cAMP in derselben Größenordnung wie Wildtyp-HsPDE2A. LAPD degradierte cGMP und cAMP bei 6- bzw. 4-facher Steigerung von vmax unter R im Vergleich zu dunkeladaptiertem Enzym. Die Aktivität von R-adaptierter LAPD wurde durch FR reduziert. Die enzymatische Aktivität und Lichtregulation von LAPD-Linkervarianten waren abhängig von der Linkerlänge. LAPD degradierte lichtabhängig cGMP in einer PDE-Reporterzelle. Dabei genügte die endogene BV-Konzentration der Säugerzelle zur Sättigung des Lichteffekts. / Genetically encoded photoreceptors in model organisms establish optogenetics. It enables non-invasive, reversible, and spatio-temporally precise perturbation of cellular and physiological signalling by light. Natural photoactivated adenylate cyclases (PACs) increase the intracellular concentration of the second messenger cyclic adenosine monophosphate (cAMP) under blue light. Hence, PACs allow the optogenetic analysis of cAMP-dependent signalling. This work complements PACs with the synthetic red-light-activated phosphodiesterase LAPD for degradation of cAMP and cyclic guanosine monophosphate (cGMP). LAPD is a chimera made up of the photosensory module of Deinococcus radiodurans bacteriophytochrome (DrBPhy) and the effector domain of cAMP/cGMP-specific H. sapiens Phosphodiesterase 2A (HsPDE2A). The fusion site was derived from the helical linkers between sensor and effector modules via structural superposition. LAPD incorporated the chromophor biliverdin (BV) after expression in E. coli and purification quantitatively, and spectrally and photochemically resembled the wildtype DrBPhy. Upon irradiation with red and far-red light (R and FR, resp.), LAPD was converted to the metastable photochemical states Pfr (far-red) and Pr (red), respectively. Fully activated LAPD catalized the hydrolysis of cGMP and cAMP with rates similar to wildtype HsPDE2A. LAPD degraded cGMP and cAMP with 6- and 4-fold increase of vmax under R, respectively, as compared to the dark state. The activity of R-adapted LAPD was reduced upon irradiation with FR. Enzymatic activity and light regulation of LAPD linker variants depended on the linker length. LAPD light-dependently degraded cGMP in a PDE reporter cell line. Endogenous BV concentrations were sufficient to saturate the light effect in the mammalian cell, which enables a true optogenetic approach.

Licht und Pflanzenhormone sind essentiell für das Wachstum und die Entwicklung von Pflanzen. Es ist nur wenig darüber bekannt, wie sie die Transkription organellärer Gene beeinflussen. In Arabidopsis thaliana gibt es drei kernkodierte Phagentyp-RNA-Polymerasen (RpoT), welche für die organelläre Transkription verantwortlich sind. Diese werden in die Plastiden (RpoTp), die Mitochondrien (RpoTm) oder zu beiden Organellen (RpoTmp) transportiert. Neben den beiden kernkodierten RNA-Polymerasen (NEP) existiert in den Plastiden eine plastidärkodierte RNA-Polymerase (PEP), welche zusätzliche Sigmafaktoren zur Promotererkennung benötigt. Um die Lichtabhängigkeit der Expression der RpoT Gene sowie NEP-transkribierter Chloroplastengene zu analysieren, wurde die Akkumulation von RpoT- und rpoB-Transkripten in 7-Tage alten Keimlingen unter verschiedenen Lichtbedingungen mittels quantitativer real-time PCR untersucht. Die Änderungen in der Transkriptakkumulation deuten darauf hin, dass rote, blaue und grüne Wellenlängen die Expression der drei RpoT Gene unterschiedlich stark stimulieren. Untersuchungen an verschiedenen Lichtrezeptor-Mutanten zeigten, dass die Lichtinduktion der RpoT Genexpression überaus komplex ist und ein interagierendes Netzwerk aus multiplen Rezeptoren und Transkriptionsfaktoren an der Signalweiterleitung beteiligt ist. Das Phytohormon Cytokinin wird durch Histidin Kinase Rezeptoren (AHK) detektiert. Es gibt drei unterschiedliche Rezeptoren: AHK2, AHK3 und AHK4. Diese sind Teil eines Zwei-Komponenten-Systems, welches Signale mit Hilfe einer Phosphorylierungskette überträgt. Der Einfluss von Cytokinin auf die plastidäre Transkription wurde mit Hilfe von Cytokininrezeptor-Mutanten untersucht, um die Funktion von AHK2, AHK3 und AHK4 zu analysieren. Um weitere Informationen darüber zu erhalten, wie die plastidäre Transkription durch PEP mittels Cytokinin reguliert wird, wurden die Hormoneffekte auf die plastidäre Transkription in Sigmafaktor-Mutanten untersucht. / Light and plant hormones are essential for plant growth and development. Only little information is available about how these signals influence the transcription of organellar genes. Arabidopsis thaliana possesses three nuclear-encoded phage-type RNA polymerases (RpoT) for organellar transcription. They are imported into plastids (RpoTp), mitochondria (RpoTm), or into both organelles (RpoTmp). Besides the two nuclear-encoded plastid polymerases (NEP), plastids contain an additional plastid-encoded RNA polymerase (PEP), which needs additional sigma factors for promoter recognition. Interested in the expression of RpoT genes and NEP-transcribed plastid genes in response to light we analyzed transcript levels of RpoT and rpoB genes in 7-day-old wild-type plants under different light conditions by quantitative real-time-PCR. The observed changes in transcript accumulation indicated that red, blue, and green light differentially stimulated the expression of all three RpoT genes. Further analyses using different photoreceptor mutants showed that light induction of RpoT gene expression is surprisingly complex based on a network of multiple photoreceptors an d downstream pathways. Cytokinin signals are perceived by the histidine kinase (AHK) receptor family. There exist three different membrane-bound receptors: AHK2, AHK3 and AHK4/CRE1. These receptors are part of a two-component signaling system which transfers signals via phosphorelay mechanisms. Interested in the potential role of AHK2, AHK3 and AHK4/CRE1 in the transduction of cytokinin signals into the chloroplast, we analyzed the influence of cytokinin on plastidial transcription in receptor mutants. To gain more information on how plastid transcription by PEP is regulated by cytokinin, the influence of cytokinin in sigma factor mutants was also studied.

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

Zusätzlich zu der eubakteriellen RNA-Polymerase (RNAP) der Plastiden sind im Zellkern von Arabidopsis thaliana drei weitere, phagentypische RNAP kodiert, die jeweils aus nur einer Einheit aufgebaut sind. Die Enzyme RpoTp und RpoTm werden in die Plastiden, bzw. die Mitochondrien transportiert, während RpoTmp in beiden Organellen zu finden ist. Um die Lichtabhängigkeit der RpoT-Gene zu untersuchen, wurde die lichtinduzierte Akkumulation ihrer Transkripte in 7-Tage alten Keimlingen, sowie 3- bzw. 9-Wochen alten Rosettenblättern mittels quantitativer real-time PCR ermittelt. Die entwicklungsabhängige Regulation der RpoT-Transkript-Akkumulation wurde außerdem während der Blattentwicklung analysiert. Zusätzlich wurde der Einfluss des circadianen Rhythmus untersucht. Es stellte sich heraus, dass die Transkriptakkumulation aller drei RpoT-Gene stark lichtinduziert war und nur marginalen circadianen Schwankungen unterlag. In weiteren Versuchen mit verschiedenen Lichtrezeptor-Mutanten und unterschiedlichen Lichtqualitäten wurde der Einfluss multipler Rezeptoren auf den Prozess der Lichtinduktion gezeigt. In den Zellen höherer Pflanzen finden sich drei Genome. Die Biogenese von Chloroplasten und Mitochondrien, sowie lebenswichtige Prozesse, wie Atmung und Photosynthese setzen oftmals die Aktivität von Genen auf mindestens zwei dieser Genome voraus. Eine intrazelluläre Kommunikation zwischen den verschiedenen Genomen ist daher unumgänglich für einen funktionierenden Stoffwechsel der Pflanze. In dieser Arbeit wurde herausgestellt, dass die Zahl mitochondrialer Genkopien in photosynthetisch inaktiven Arabidopsis-Keimlingen drastisch erhöht ist. Bei der Untersuchung des DNA-Gehaltes in Proben, die Altersstufen von 2-Tage alten Keimblättern bis hin zu 37-Tage alten, seneszenten Rosettenblättern umfassten, fand sich ein deutlicher Anstieg der Kopienzahlen in älteren Rosettenblättern. Außerdem unterschieden sich die Kopienzahlen der untersuchten Gene zum Teil erheblich voneinander. / In addition to eubacterial-like multi-subunit RNA polymerases (RNAP) localized in plastids and the nucleus, Arabidopsis thaliana contains three phage-like single-unit, nuclear-encoded, organellar RNAPs. The enzymes RpoTp and RpoTm are imported into plastids and mitochondria, respectively, whereas RpoTmp shows dual targeting properties into both organelles. To investigate if expression of the RpoT genes is light-dependent, light-induced transcript accumulation of RpoTm, RpoTp and RpoTmp was analyzed using quantitative real-time-PCR in 7-day-old seedlings as well as in 3- and 9-week-old rosette leaves. To address the question whether RpoT transcript accumulation is regulated differentially during plant development transcript abundance was measured during leaf development. Additionally, effects of the plants circadian rhythm on RpoT transcript accumulation were analyzed. Transcripts of all three RpoT genes were found to be strongly light-induced even in senescent leaves and only marginally influenced by the circadian clock. Further analyses employing different photoreceptor mutants and light qualities revealed the involvement of multiple receptors in the light-induction process. The biogenesis of mitochondria and chloroplasts as well as processes like respiration and photosynthesis require the activity of genes residing in at least two distinct genomes. There have to be ways of intracellular communication between different genomes to control gene activities in response to developmental and metabolic needs of the plant. In this study, it was shown that gene copy numbers drastically increased in photosynthetically inactive Arabidopsis seedlings. Mitochondrial DNA contents in cotyledons and leaves ranging in age from 2-day-old cotyledons to 37-day-old senescent rosette leaves were examined. A common increase in senescing rosette leaves and drastic differences between individual genes were found, revealing the importance of an integrative chondriome in higher plant cells.

Sensorische Photorezeptorproteine vermitteln vielfältige Lichtreaktionen in allen Domänen des Lebens. Oftmals dienen verschiedene, durch helikale ‚Linker’ gekoppelte, Module der Lichtperzeption (Sensor) und der Umwandlung in ein biologisches Aktivität (Effektor). Der Zusammenbau chimärer Photorezeptoren aus unterschiedlichen Sensoren und Effektoren ermöglicht die präzise und minimalinvasive Regulation zellulärer Signalwege mit Hilfe von Licht, zu therapeutischen oder analytischen Zwecken. Eine große Herausforderung stellt dabei die korrekte Fusion der Linker beider Module dar, die Kommunikation zwischen Sensor und Effektor erlaubt. Die vorliegende Arbeit nimmt sich diesem Problem an und untersucht Strategien zum effizienten Bau chimärer Photorezeptorproteine. Ein rationaler, auf Sequenz- und Strukturhomologie der parentalen Proteine basierender Ansatz wurde maßgeblich durch unzureichendes Verständnis der funktionellen Mechanismen dieser modularen Proteinen erschwert. Die neuentwickelte und PATCHY-Methode umgeht dieses Hindernis, indem sie eine Bibliothek von Chimären aller Kombinationen der parentalen Linker generiert, welche anschließend mittels bakterieller Testsysteme nach funktionalen Varianten durchsucht wird. Angewendet auf die Fusion eines LOV-Blaulichtsensors und eines Histidinkinase-Effektors fanden sich sowohl lichtaktivierte, als auch zu lichtreprimierte Chimären, deren Linkerlängen jeweils einer Heptadenperiodizität folgten. Dass weniger als 5% aller Linkerkombinationen zu lichtregulierten Chimären führten, deutet zudem auf eine feine Abstimmung von Linkersequenz und Proteinfunktion hin. Die systematische Analyse von Fusionsvarianten mit PATCHY dient daher nicht nur der Entwicklung chimärer Rezeptorproteine zur Manipulation zellulärer Prozesse. Sondern sie zeigt darüber hinaus, komplementär zum rationalen Ansatz, molekulare Faktoren auf, die zur Modulkompatibilität und Signaltransduktion modularer Rezeptorproteine beitragen. / Sensory photoreceptor proteins mediate diverse responses to ambient light in all domains of life. Often distinct modules coupled by helical linkers enable light perception (sensor) and biological output function (effector). Rewiring different sensor and effector modules into photoreceptor chimeras allows using light to control target cellular processes with high spatiotemporal accuracy and minimal invasiveness for therapeutic or analytical purposes. Thereby, a major design challenge is fusing the linkers from both modules in a way that preserves signal transduction within the chimera. The present study tackles this issue and explores strategies for engineering photoreceptor chimeras. An initial rational-design approach guided by sequence and structure homology of the parent proteins was greatly hampered by insufficient knowledge of signaling mechanisms within these modular proteins. A novel and easy-to-use brute-force strategy, termed PATCHY (primer-aided truncation for the creation of hybrid enzymes) circumvents this problem by generating a complete library of fusion variants between target modules harboring all combinations of the parent linkers. Screening fusion libraries of a LOV (light-oxygen-voltage) blue-light sensor coupled to a histidine-kinase effector yielded light-induced and light-repressed chimeras, each group complying with a heptad periodicity of linker lengths. With less than 5% of all possible variants exhibiting light regulation, a delicate fine-tuning of linker sequence and protein function became evident. Thus, systematic testing of fusion variants with PATCHY not only facilitates the development of photoreceptor chimeras for manipulating cellular processes. Complementary to rational design, it also reveals molecular cues determining module compatibility and signal transduction in modular signal receptors.

Die Modifikation von Genen ist in den molekularen Biowissenschaften ein fundamentales Werkzeug, um die Funktion von Genen zu studieren (Reverse Genetik). Diese Arbeit hat erfolgreich Zinkfinger- und CRISPR/Cas9-Nukleasen für die Verwendung in C. reinhardtii etabliert, um Gene im Kerngenom gezielt auszuschalten und präzise zu verändern. Basierend auf vorausgegangener Arbeit mit Zinkfingernukleasen (ZFN) konnte die Transformationseffizienz um das 300-fache verbessert werden, was die Inaktivierung von Genen auch in motilen Wildtyp-Zellen ermöglichte. Damit war es möglich, die Gene für das Kanalrhodopsin-1 (ChR1), Kanalrhodopsin-2 (ChR2) und das Chlamyopsin-1/2-Gen (COP1/2) einzeln und gemeinsam auszuschalten. Eine Analyse der Phototaxis in diesen Stämmen ergab, dass die Phototaxis durch Inaktivierung von ChR1 stärker beeinträchtigt ist als durch Inaktivierung von ChR2. Um das CRISPR/Cas9-System zu verwenden, wurden die Transformationsbedingungen so angepasst und optimiert, dass der Cas9-gRNA-Komplex als in vitro hergestelltes Ribonukleoprotein in die Zellen transformiert wurde. Um die Bedingungen für präzise Genmodifikationen zu messen und zu verbessern, wurde das SNRK2.2-Gen als Reportergen für eine „Blau-Grün Test“ etabliert. Kleine Insertionen von bis zu 30 bp konnten mit kurzen Oligonukleotiden eingefügt werden, während größere Reportergene (mVenus, SNAP-Tag) mithilfe eines Donor-Plasmids generiert wurden. In dieser Arbeit konnten mehr als 20 nicht-selektierbare Gene – darunter 10 der 15 potenziellen Photorezeptorgene – mit einer durchschnittlichen Mutationsrate von 12,1 % inaktiviert werden. Insgesamt zeigt diese Arbeit in umfassender Weise, wie Gen-Inaktivierungen und Modifikationen mithilfe von ZFNs und des CRISPR/Cas9-Systems in der Grünalge C. reinhardtii durchgeführt werden können. Außerdem bietet die Sammlung der zehn Photorezeptor-Knockouts eine aussichtsreiche Grundlage, um die Vielfalt der Photorezeptoren in C. reinhardtii zu erforschen. / Gene editing is a fundamental tool in molecular biosciences in order to study the function of genes (reverse genetics). This study established zinc-finger and CRISPR/Cas9 nucleases for gene editing to target and inactivate the photoreceptor genes in C. reinhardtii. In continuation of previous work with designer zinc-finger nucleases (ZFN), the transformation efficiency could be improved 300-fold, which enabled the inactivation of genes in motile wild type cells. This made it possible to disrupt the Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2) and Chlamyopsin-1/2 (COP1/2) genes individually and in parallel. Phototaxis experiments in these strains revealed that the inactivation of ChR1 had a greater effect on phototaxis than the inactivation of ChR2. To apply the CRISPR/Cas9 system, the transformation conditions were adapted and optimized so that the Cas9-gRNA complex was successfully electroporated into the cells as an in vitro synthesized ribonucleoprotein. This approach enabled gene inactivations with CRISPR/Cas9 in C. reinhardtii. In order to measure and improve the conditions for precise gene modifications, the SNRK2.2 gene was established as a reporter gene for a ‘Blue-Green test’. Small insertions of up to 30 bp were inserted using short oligonucleotides, while larger reporter genes (mVenus, SNAP-tag) were integrated using donor plasmids. Throughout this study, more than 20 non-selectable genes were disrupted, including 10 of the photoreceptor genes, with an average mutation rate of 12,1 %. Overall, this work shows in a comprehensive way how gene inactivations and modifications can be performed in green alga C. reinhardtii using ZFNs or CRISPR/Cas9. In addition, the collection of the ten photoreceptor knockouts provides a promising source to investigate the diversity of photoreceptor genes in C. reinhardtii.